The utilization of cancer-specific antigens and molecular markers in the diagnosis and treatment of malignant tumors is a goal of medical professionals. The realization of this goal has been advanced by the use of in vivo animal and in-vitro model systems in order to map out the relevant steps of a cancer-specific immune response and also the steps required for its use in cancer therapy. Methods which utilize cancer-specific and/or cancer-associated markers for diagnosis and therapy have been reported, but the principal shortcoming preventing the implementation of these methods has been the paucity of cancer-specific or highly cancer-associated antigens and other markers of cancer in humans.
Some progress in obtaining candidates for cancer-specific or highly cancer associated antigens for cancer diagnosis and treatment includes the construction of synthetic peptides, for example, for the production of antibodies specific for the peptides, where the peptides are potentially useful as markers. For example, different epitopes have been found to be associated with mucins from malignant cells, in contrast to mucins in non-malignant cells. Aberrant glycosylation has been found in some peptides from tumors.
Vaccines and immunotherapies using specific domains of membrane proteins have been reported to be more effective than vaccines and immunotherapy using entire glycoproteins.
At present, not enough cancer antigens or markers are available for use in implementing robust cancer diagnostic or therapeutic methods in humans. Human cancer antigens and markers described to date are either inadequate or too few in number to provide useful clinical tools.
For example, the MAGE family of antigens described by Boon et al. (1994) are reported to be cancer-associated antigens. Cancer-associated antigens are those expressed in greater quantity in molecules in or on, or derived from cancer cells, but are also concurrently expressed in molecules from normal cells. This duality complicates therapeutic utility of the antigens for vaccines and antibodies where positive effects are dependent upon reaching a therapeutic dose before a toxic dose level is realized. Other limitations of the MAGE antigens are that they are also intracellular cancer antigens thus greatly diminishing their utility for cancer cell targeting which is more effective for cell surface antigens. Intracellular antigens serve as poor localizing targets for immunotherapy, targeted cytotoxic therapeutic agents, cell receptor blocking agents, other cell-surface disruptive agents, and for diagnostic imaging. They are poor immunogenic targets for eliciting a measurable immune response. Their release for direct quantification is unpredictable because cancer cell disruption is required.
Cheever et al.(1997a,b) have described the potential diagnostic and therapeutic use of oncogenic proteins which are expressed by both cancer and normal cells. They describe using oncogenic proteins with site-specific mutations as the cancer-specific antigens. However, the oncogenic proteins cited by Cheever, designated the p21 proteins, are intracellular and thus share the drawbacks of other intracellular antigens, that is, cannot the detected on cell surfaces. Furthermore, mutated expression is not always manifested by expressed oncogenic proteins in all cancer cells, thus leaving some cells to express oncogenic proteins which are subject to self-recognition and are thus poorly immunogenic.
Cheever's other example, the erbB-2 epidermal growth factor receptor, also known as HER-2/neu, is used to support the hypothesis that breaking self-recognition offers a novel therapeutic pathway (Disis et al., 1998a, b; 1999) although that method is not commonly accepted by most immunologists. The erbB-2 molecule is a transmembrane receptor with a significant extracellular portion. Its extracellular domain is commonly believed to be structurally similar for both cancer cells and normal cells. Thus, the advantages it possesses over intracellular antigen candidates is minimized because of its susceptibility to down regulation of any specific immune response on the basis of self recognition.
Use of derivatives of bombesin, an amphibian protein, was an attempt to inhibit growth of tumor cells that respond to bombesin (Knight et al., 1997). Bogden and Moreau attempted to treat human cancer by administering analogs of a biologically active peptide to a patient. However, these attempts used molecules that did not differentiate normal from cancer cells.
The deglycosylated mucins described by Barratt et al., 1998 and Henderson et al., 1998 are another example of a class of cancer-associated antigens with epitopes detectable outside of the cell. Mucins are large secreted and/or transmembrane glycoproteins with greater than 50% of their molecular weight derived from O-linked carbohydrates attached to serine and theonine. Their cancer specificity depends on a greater degree of altered structure rather than on numerical over-expression. The loss or diminution of carbohydrate side chains emanating from a central core protein makes the Muc proteins more immunogenic. Finn et al. ascribes this immunogenity as a result of significant altered molecular folding made possible by a release from molecular rigidity conferred by the many projecting glycoside chains found in mucin molecules in non-cancerous cells. The alteration in folding creates neo-epitopes which help break immune self-recognition and also separately facilitates stimulation of a cellular immune response. Problems with the Muc antigens include insufficient diversity needed to provide wide enough antigenic coverage for many cancers, and their rapid cellular release rate as a consequence of Muc antigens being secreted proteins, as opposed to functional cell membrane proteins such as receptor molecules, receptor-like molecules, or cell adhesion molecules. The latter attribute makes Muc antigens less effective therapeutic and imaging targets.
Hudziak et al. (1998a, b) describes the therapeutic utility of monoclonal antibodies specific for the extracellular domain of the normal HER-2/neu receptor (also known as erbB-2). The basis of this therapeutic method is described as the inhibition of the cancer-proliferative function of the receptor caused by the binding of a specific monoclonal antibody to the outer domain of the receptor thereby preventing the binding of circulating epidermal growth factor and other ligands to the receptor. Decreased or absent growth factor stimulation results in cancer cell death through apoptosis. This method relies on higher expression of Her-2/neu on cancer cells as compared to normal cells. Therapy is dose dependent. Sufficient blocking antibody must be administered so as to block enough cancer cell HER-2/neu molecules required to affect cancer cell death without causing normal cell death or normal cell toxicity. Adequate therapeutic dosing is not possible for all patients who express HER-2/neu on the their tumor cells. Some cancer patients express adequate amounts of HER-2/neu; some express low amounts; and yet others express none. Consequently, this therapeutic method works marginally, or not at all for most patients. Occasionally, when patient circumstances are appropriate, this method is capable of affecting total cancer remission. This limited result illustrates the basic soundness of a therapeutic method provided that a large repertoire of cancer-specific or cancer-associated functional targets were made available. However, more and better cancer-specific and cancer-associated antigens are needed to make these approaches clinically useful.
A method of preparing phosphorylated tumor specific peptides was reported by Calenoff (1998).
There are suggestions of expression of cancer-specific or cancer-associated molecules, as well as over-expression or under-expression of the molecules in or on cancer cells. For example, many receptor-like adhesion proteins found on the surface of cells have been described. Some of these adhesion proteins are reported to facilitate tumor migration and invasion (Zheng et al., 1999; Rabinovitz et al., 1995; Friedl et al., 1998) or metastatic spread (Romanov et al., 1999). Others are reported to facilitate essential functioning for both cancer cells and tissues and for normal cells and tissues (Ekblom et al, 1998; Fleischmajer et al., 1998; Bonkoff, 1998; Fujiwara et al., 1998; Lohi, 1998). Blocking certain functions facilitated by receptor-like adhesion molecules is suggested to provide new therapeutic modalities for eradicating or controlling cancer (Ruoslahti et al., 1997). Although various adhesion molecule isotypes are reported to be over-expressed (Damiano et al., 1999; Liapis et al., 1996; Begum et al., 1995; Katsura et al., 1998) or underexpressed (Furakawa et al., 1994; Damjanovich et al., 1997; Luguki et al., 1999) on cancer cells as compared to normal cells, none have been described which possess the cancer-specific or highly cancer-associated structural modifications of the present invention.